Human immunodeficiency virus (HIV) is a retrovirus that infects cells those possess the CD4 receptor. This infection causes the depletion of CD4 positive lymphocytes, which is a major clinical finding in progressive infection. In HIV infection, the absolute number of CD4 positive lymphocytes is an important marker for prognosis, classification of the state of disease, treatment decision and monitoring of therapy. Immunophenotyping using lysed whole blood stained with monoclonal antibody panels and analyzed by flow cytometry is the current standard method for determination of CD4 positive lymphocytes.
Recently several types of immunophenotyping reagents for measurement of CD4 positive lymphocytes have been developed and routinely used in clinical laboratory. The three-color immunophenotyping reagent is a common flow cytometric reagent for determining CD4 positive lymphocytes in routine laboratory. The available three-color reagents contain peridinin chlorophyll protein (PerCP) or phycoerythrincyanin 5.1 (PE-Cy5) labeled CD45 monoclonal antibody for discrimination of white blood cells (leucocytes) and red blood cells (erythrocytes) and contain FITC labeled CD3 monoclonal antibody and PE labeled CD4 monoclonal antibody for enumeration of CD4 positive lymphocytes. Unlike two-color reagent, a disadvantage of three-color reagents is that it cannot indicate number of monocytes that are contaminated in the lymphocyte population gated during flow cytometric measurement. If significant numbers of the contaminated monocytes are presented in the acquisition lymphocyte gate, the obtained CD4 positive lymphocyte number will be erroneously decreased.
We have developed a three-color reagent that can enumerate percentage of CD4 positive lymphocytes in lymphocyte population and can determine the contamination of monocytes in the acquisition lymphocyte gate, simultaneously. This reagent contains 7-aminoactinomycin D (7-AAD) which can intercalate into double stranded DNA of white blood cells and allow white blood cells, but not red blood cells, to be detected with FL3 detector of flow cytometer. The reagent also contains FITC labeled CD4 monoclonal antibody and PE labeled CD14 monoclonal antibody, which allow CD4 positive cells and monocytes to be detected with FL1 and FL2 detector, respectively.